It is known that a large number of organisms are possessed of a mechanism in their bodies, which generates circadian rhythm with ablout 24 hour cycle (Reference 1). In mammals, the mechanism generating circadian rhythm controls sleep-wake rhythm, blood pressure, body temperature and a part of hormone secretion rhythm (Reference 2, Reference 3). As diseases caused by the circadian rhythm disturbance, sleep-wake rhythm disorder (delayed sleep phase syndrome (DSPS), non-24 hour sleep-wake syndrome), seasonal depression, jet lag syndrome (JET-LAG), sleep disturbance in night and day shift workers, nocturnal poriomania and delirium found in patients with senile dementia and the like have been reported (References 4 to 7). In addition, there is a report stating that a part of children with school refusal or workers with refusal to attend firm as a social problem is caused by a circadian rhythm disorder (Reference 8). Increase in the number of patients with rhythm disorder is expected in the future by the increase in advanced aged and the progress of globalization of social structure, but it is the present situation that a secure rhythm disorder improving agent is not present. On the other hand, there are reports stating that bright light therapy, including staring at a light of about 5,000 luxes continuously for several hours in the early morning, shows excellent therapeutic effect on nocturnal poriomania and delirium in patients with senile dementia, rhythm disturbance in patients with delayed sleep phase syndrome (DSPS) or the like (References 6, 7 and 9 to 12). However, since the bright light therapy which requires looking at a high lux light source for a prolonged period of time is painful or burden for patients and their caretakers, agents as substitutes for this light therapy are highly expected.
Based on tissue destruction and tissue transplantation experiments, it has been found in 1972 that the rhythm center of mammals is present in suprachiasmatic nucleus (SCN) (References 13 and 14). However, molecular mechanism of the rhythm generation has been unclear until recent years (Reference 15). On the other hand, an arrhythmic mutant (Period mutant) of Drosophila melanogaster has been prepared by a genetic technique and then a Period gene of Drosophila melanogaster has been cloned (Reference 16, Reference 17). Since oscillatory expression of Period gene with about 24 hour cycle is achieved in Drosophila melanogaster through the migration of translated Period protein (PERIOD) into the nucleus and subsequent inhibition of its own transcription (negative feedback mechanism), it is considered that outputs of circadian rhythm (behavior, timing of eclosion) are finally developed by this (References 18 and 19). In mammals, on the other hand, human and mouse Period1 gene (Period1; Per1) has been cloned in 1997 as a homologue of the Drosophila Period gene (Reference 20, Reference 21). Thereafter, mouse Period2 gene (Period2; Per2) (References 22 and 23) and mouse Period3 gene (Period3; Per3) (References 24 and 25) have been cloned. In addition to these, mouse Clock gene (Clock) (Reference 26) and mouse Bmal1 gene (Bmal1) (Reference 27) have been reported as mammalian clock genes. Thus, it is possible for now to understand the rhythm generation mechanism at the molecular level. Actually, it has been found that the Clock gene and Bmal1 gene are important for the circadian oscillation of a clock gene (References 44 and 45) and that a protein encoded by the Clock gene and a protein encoded by the Bmal1 gene bind to a CACGTG type E-box and activate transcription of said clock gene, namely, the CACGTG type E-box sequence is essential for the transcriptional activation by the CLOCK and BMAL1 (Reference 27).
A transgenic rat (mPer1; luc transgenic rat) harboring a DNA prepared by ligating an upstream sequence of a mouse clock gene, Period1 gene (mPer 1), with a luciferase gene has been reported in 2000 (Reference 28). It has been reported that the Period1 shows oscillatory expressions in not only the suprachiasmatic nucleus but also in peripheral tissues of the living body by measuring the luciferase activity in real time using a photomultiplier tube detector (Photomal) (Reference 28). In addition, similar results have been reported also on a transgenic mouse (mPer1; luc transgenic mouse) harboring a DNA prepared by ligating an upstream sequence of the Period1 gene with a luciferase gene (Reference 43).
It has been suggested that the Period2 gene among the three Period gene homologues takes an important role in the rhythm generation, because its circadian rhythm disappears in mutant mice with artificial gene mutation (Reference 29). Also, it has been reported that the cause of a familial advanced sleep phase syndrome (ASPS) is a point mutation of the Period2 gene (Reference 30). Thus, the Period2 gene is a gene which not only shows abnormal rhythm in the mutant mice but also relates to rhythm disorders in human has been confirmed.
Only the one report regarding the upstream region of Period2 gene is WO 01/07654 on a mouse sequence, and said international publication describes a DNA sequence by defining it as a sequence which controls mouse Period2 transcription and describes about a method for identifying a Period2 transcription inhibitor, which comprises supplying a cell containing sequence (which controls Per2 transcription)-linked reporter gene, introducing a test compound and assaying transcription of the reporter gene. However, there is no specific example on actually obtaining the above DNA sequence described as a sequence which controls mouse Period2 transcription, and there is no description such that it can be obtained. Also, there is no specific example on the determination of transcription start site or measurement of transcriptional activity, too. In addition, there are positions in the disclosed DNA sequence where bases cannot be specified, and sequence information regarding upstream sequence of the Period2 gene is not specifically disclosed.
Also, there are many reports on the analysis of the upstream sequence of Period1 gene, but since Period1 and Period2 genes are located in different chromosomes and have no characteristic common sequence, it did not become information for deducing the Period2 promoter sequence.
Great concern has been directed toward the development of a tool for the screening of useful substances as rhythm disorder improving agents having a mechanism of function to control expression of biological clock genes and a method for screening substances capable of controlling expression of biological clock genes.